The dynamics of microtubules is regulated mainly by microtubule-associated proteins (MAPs) and plays an important role in plant development and response to environmental signals. End-binding protein 1 (EB1) is a MAP specially binding to the microtubule plus end. Blast search of tomato genome showed two EB1 genes, which were named as SlEB1a (Solyc03g116370) and SlEB1b (Solyc02g092950) in this study. Transgenic tomato plants over-expressing SlEB1a or RNA interfering both SlEB1a and SlEB1b were constructed, and their sensitivity to microtubule depolymerization drug propyzamide and salt stress were analyzed. In this study, we determined the role of tomato EB1 (SlEB1) in the response to salt stress. Compared to the wild-type control plants, OE plants were more sensitive to 1 μmol/L propyzamide, whereas RNAi plants were more tolerant to 1 μmol/L propyzamide; in contrast, OE plants were more tolerant to 100 mmol/L NaCl, whereas RNAi plants were more sensitive to 100 mmol/L NaCl. Thus, SlEB1 might positively regulate salt stress response by negatively regulating the dynamics of tomato cortical microtubules. This study forms a basis for how cortical microtubule dynamics plays a role in plant response to salt stress.